With the discovery of new biomarkers associated with tumor development, many of these tumor-associated antigens (TAA) are being utilized in immunotherapeutic modalities designed to induce anti-tumor directed cytotoxic immune responses. It is increasingly clear that not any one of these TAA is sufficient, as a single entity to develop an immunotherapeutic treatment agent. Consequently, our efforts are being focused on developing multi-targeted immunotherapeutic approach against TAA. The overall goal of the current project is to expand our immunotherapeutic approach for the treatment of metastatic colorectal cancer (mCRC) using a multiple targeted approach. Drs. Jeffery Schlom and James Gully at the National Cancer Institute have agreed to collaborate with us to reach this goal. We have achieved safety, dose response, and an increased overall survival in mCRC patients using our Ad5 [E1-, E2b-]-CEA(6D) (ETBX-011) immunotherapeutic as a single agent. We believe that adding an anti-cancer stem cell (CSC) approach can lead to increased efficacy particularly against tumor metastases. Thus, we have added Brachyury as a TAA CSC target. Brachyury represents a relatively new but attractive target for immunotherapy. It is a member of the T-box family of transcription factors that play key roles during early development, mostly in the formation and differentiation of normal mesoderm and is characterized by a highly conserved DNA-binding domain designated as T-domain. Recently, epithelial-mesenchymal transition (EMT) has been recognized as a key step during the progression of primary tumors into a metastatic state in which Brachyury plays a crucial role. Tumor EMT has been demonstrated to be associated with the acquisition of CSC-like features that includes acquisition and maintenance of CSC-like characteristics, resistance to conventional therapeutics, chemotherapy and radiation, and to some small-molecule targeted therapies. Over-expression of Brachyury in human carcinoma cells induces changes characteristic of EMT, including up-regulation of mesenchymal markers, down-regulation of epithelial markers, and an increase in cell migration and invasion. Importantly, Brachyury is immunogenic and Brachyury-specific CD8+ T cells expanded in vitro can lyse Brachyury expressing tumor cells. These features make it one of the more attractive TAA that can be utilized broadly for immunotherapeutic purposes but especially for mCRC. Brachyury is incorporated into our recombinant Ad5 [E1-, E2b-] platform for use in the multiple immunotherapy approach to treat mCRC. We believe that this platform will enable multiple immunizations that will functionally boost the immune response to this target and may prevent metastases. We will incorporate Brachyury into our recombinant Ad5 [E1-, E2b-] platform to produce a new and more potent immunotherapeutic for mCRC that will target primary and mCRC stem cells. We believe these two TAA targets as a single delivery immunotherapeutic agent, will enhance the clinical responses of our Ad5 [E1-, E2b-]- CEA(6D) approach by targeting CSC and will result in an increased overall survival of patients with mCRC.